This invention relates to polypeptide hormone analogues that exhibit enhanced pharmaceutical properties, such as more rapid pharmacokinetics at polypeptide concentrations greater than are ordinarily employed in pharmaceutical formulations. This invention also relates to insulin analogues that are modified by the incorporation of non-standard amino acids to enable their formulation at concentrations higher than 100 units per ml (U-100) such that (i) rapid-acting pharmacokinetic (PK) and pharmacodynamic (PD) properties are retained relative to wild-type human insulin at a U-100 concentration and such that (ii) their mitogenic properties are not elevated relative to wild-type human insulin. Such non-standard sequences may optionally contain standard amino-acid substitutions at other sites in the A or B chains of an insulin analogue.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. An example of a medical benefit would be optimization of the pharmacokinetic properties of a protein. An example of a further societal benefit would be the engineering of proteins amenable to formulation at high protein concentrations with deterioration of the PK/PD properties of the formulation. An example of a therapeutic protein is provided by insulin. Analogues of insulin containing non-standard amino-acid substitutions may in principle exhibit superior properties with respect to PK/PD or the dependence of PK/PD on the concentration of insulin in the formulation. The challenge posed by the pharmacokinetics of insulin absorption following subcutaneous injection affects the ability of patients with diabetes mellitus (DM) to achieve tight glycemic control and constrains the safety and performance of insulin pumps.
A particular medical need is posed by the marked resistance to insulin exhibited by certain patients with DM associated with obesity, by certain patients with DM associated with a genetic predisposition to insulin resistance, and by patients with DM secondary to lipodystrophy, treatment with corticosteroids, or over-secretion of endogenous corticosteroids (Cushing's Syndrome). The number of patients with marked insulin resistance is growing due to the obesity pandemic in the developed and developing worlds (leading to the syndrome of “diabesity”) and due to the increasing recognition of a monogenic form of DM arising from a mutation in mitochondrial DNA in which insulin resistance can be unusually severe (van den Ouweland, J. M., Lemkes, H. H., Ruitenbeek, W., Sandkuijl, L. A., de Vijlder, M. F., Struyvenberg, P. A., van de Kamp, J. J., & Maassen, J. A. (1992) Mutation in mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nature Genet. 1, 368-71). Treatment of such otherwise diverse subsets of patients typically requires the subcutaneous injection of large volumes of regular insulin formulations (U-100 strength; ordinarily 0.6 mM insulin or insulin analogue). Injection of such large volumes can lead to pain and variability in the rate of onset and duration of insulin action. Although a U-500 formulation of wild-type insulin is available for clinical use (Humulin® R U-500; Eli Lilly and Co.), the increase in insulin concentration from 0.6 mM to 3 mM leads to a delay in the onset, and prolongation, of insulin action such that the PK/PD properties of Humulin® R U-500, or similar such products, resemble those of a micro-crystalline suspension of protamine-zinc-containing insulin hexamers; this formulation has long been designated neutral protamine Hagadorn (NPH). Prandial use of a U-500 formulation of wild-type insulin by subcutaneous injection would thus be expected to decrease the efficacy of glycemic control and increase the risk of hypoglycemic episodes. Use of Humulin® R U-500, or similar such products in a device for continuous subcutaneous insulin infusion (CSII; an “insulin pump”) would likewise be expected to interfere with the ability of the patient or control algorithm to make effective adjustments in insulin infusion rates based on current or past measurements of blood glucose concentrations, leading to suboptimal glycemic control and increased risk of hypoglycemic events.
A well-established principle of insulin pharmacology relates the aggregation state of the injected insulin molecule to the time course of absorption from the depot into capillaries and hence into the systemic circulation. In general the more aggregated are the insulin molecules into high-molecular weight complexes, the greater the delay in absorption and more prolonged the insulin action. Amino-acid substitutions in the insulin molecule that weaken its self-assembly are known in the art to be associated with more rapid absorption relative to wild-type human insulin; examples are provided by the substitution ProB28→Asp (insulin aspart, the active component of Novolog®; Novo-Nordisk, Ltd) and by the paired substitutions ProB28→Lys and LysB29→Pro (insulin Lispro, the active component of Humalog®; Eli Lilly and Co.). Conversely, amino-acid extensions or chemical modifications of the insulin molecule that cause a shift in its isoelectric point (pI) from ca. pH 5 to ca. pH 7 are known in the art to lead to isoelectric precipitation of the modified insulin in the subcutaneous depot; such high molecular-weight complexes provide prolonged absorption as a basal insulin formulation. Examples are provided by NovoSol Basal® (a discontinued product of Novo-Nordisk in which ThrB27 was substituted by Arg and in which the C-terminal carboxylate moiety of ThrB30 was amidated) and insulin glargine (the active component of Lantus®, a basal formulation in which the B chain was extended by the dipeptide ArgB31-ArgB32; Sanofi-Aventis, Ltd.). (NovoSol Basal® and Lantus® each contain the additional substitution AsnA21→Gly to enable their soluble formulation under acidic conditions (pH 3 and pH 4 respectively) without chemical degradation due to deamidation of AsnA21.) Prolongation of classical micro-crystalline insulin suspensions (NPH, semi-lente, lente, and ultra-lente) exhibit a range of intermediate-to-long-acting PK/PD properties reflecting the respective physico-chemical properties of these micro-crystals and their relative rates of dissolution.
The above insulin products, including current and past formulations of wild-type human insulin or animal insulins, employ or employed self-assembly of the insulin molecule as a means to achieve chemical stability, as a means to avoid fibril formation, as a means to modulate PK/PD properties, or as a means to achieve a combination of these objectives. Yet insulin self-assembly can also introduce unfavorable or undesired properties. The non-optimal prolonged PK/PD properties of Humulin® R U-500 (or a similar such product), for example, are likely to be the result of hexamer-hexamer associations in the formulation and in the subcutaneous depot. Indeed, studies of wild-type bovine insulin zinc hexamers in vitro by laser light scattering have provided evidence of progressive hexamer-hexamer interactions in the concentration range 0.3-3 mM. Current and past strategies for the composition of insulin formulations and design of insulin analogues therefore face and have faced an irreconcilable barrier to the development of a rapid-acting ultra-concentrated insulin formulation: whereas self-assembly is necessary to obtain chemical and physical stability, its progressive nature above 0.6 mM leads to unfavorable prolongation of PK/PD.
During the past decade specific chemical modifications to the insulin molecule have been described that selectively modify one or another particular property of the protein to facilitate an application of interest. Whereas at the beginning of the recombinant DNA era (1980) wild-type human insulin was envisaged as being optimal for use in diverse therapeutic contexts, the broad clinical use of insulin analogues in the past decade suggests that a suite of non-standard analogs, each tailored to address a specific unmet need, would provide significant medical and societal benefits. Substitution of one natural amino acid at a specific position in a protein by another natural amino acid is well known in the art and is herein designated a standard substitution. Non-standard substitutions in insulin offer the prospect of accelerated absorption without worsening of PK/PD as a function of insulin analogue concentration in the range 0.6-3.0 mM.
Administration of insulin has long been established as a treatment for diabetes mellitus. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). The sequence of insulin is shown in schematic form in FIG. 1. Individual residues are indicated by the identity of the amino acid (typically using a standard three-letter code), the chain and sequence position (typically as a superscript).
Aromatic side chains in insulin, as in globular proteins in general, may engage in a variety of hydrophobic and weakly polar interactions, involving not only neighboring aromatic rings but also other sources of positive- or negative electrostatic potential. Examples include main-chain carbonyl- and amide groups in peptide bonds. Hydrophobic packing of aromatic side chains is believed to occur within the core of proteins and at non-polar interfaces between proteins. Such aromatic side chains can be conserved among vertebrate proteins, reflecting their key contributions to structure or function. An example of a natural aromatic amino acid is phenylalanine. Its aromatic ring system contains six carbons arranged as a planar hexagon. Aromaticity is a collective property of the binding arrangement among these six carbons, leading to π electronic orbitals above and below the plane of the ring. These faces exhibit a partial negative electrostatic potential whereas the edge of the ring, containing five C—H moieties, exhibits a partial positive electrostatic potential. This asymmetric distribution of partial charges gives rise to a quadrapole electrostatic moment and may participate in weakly polar interactions with other formal or partial charges in a protein. An additional characteristic feature of an aromatic side chains is its volume. Determinants of this volume include the topographic contours of its five C—H moieties at the edges of the planar ring. Substitution of one C—H moiety by a C—F moiety would be expected to preserve its aromaticity but introduced a significant dipole moment in the ring due to the electronegativity of the fluorine atom and consequent distortion of the π electronic orbitals above and below the plane of the ring. Whereas the size of the C—F moiety is similar to that of the native C—H moiety (and so could in principle be accommodated in diverse protein environments), its local electronegativity and ring-specific fluorine-induced electrostatic dipole moment could introduce favorable or unfavorable electrostatic interactions with neighboring groups in a protein. Examples of such neighboring groups include, but are not restricted to, CO—NH peptide bond units, lone pair electrons of sulfur atoms in disulfide bridges, side-chain carboxamide functions (Asn and Gln), other aromatic rings (Phe, Tyr, Trp, and His), and the formal positive and negative charges of acidic side chains (Asp and Glu), basic side chains (Lys and Arg), a titratable side chain with potential pKa in the range used in insulin formations (His), titratable N- and C-terminal chain termini, bound metal ions (such as Zn2+ or Ca2+), and protein-bound water molecules.
An example of a conserved aromatic residue in a therapeutic protein is provided by phenylalanine at position B24 of the B chain of insulin (designated PheB24). This is one of three phenylalanine residues in insulin (positions B1, B24, and B25). A structurally similar tyrosine is at position B26. The structural environment of PheB24 in an insulin monomer is shown in a ribbon model (FIG. 2A) and in a space-filling model (FIG. 2B). Conserved among vertebrate insulins and insulin-like growth factors, the aromatic ring of PheB24 packs against (but not within) the hydrophobic core to stabilize the super-secondary structure of the B chain. PheB24 lies at the classical receptor-binding surface and has been proposed to direct a change in conformation on receptor binding. PheB24 packs at the dimer interface of insulin and so at three interfaces of an insulin hexamer. Its structural environment in the insulin monomer differs from its structural environment at these interfaces. In particular, the surrounding volume available to the side chain of PheB24 is larger in the monomer than in the dimer or hexamer.
A major goal of insulin replacement therapy in patients with DM is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinaphthy, blindness, and renal failure. Because the pharmacokinetics of absorption of wild-type human insulin or human insulin analogues—when formulated at strengths greater than U-100—is often too slow, too prolonged and too variable relative to the physiological requirements of post-prandial metabolic homeostasis, patients with DM associated with marked insulin resistance often fail to achieve optimal glycemic targets and are thus at increased risk of both immediate and long-term complications. Thus, the safety, efficacy, and real-world convenience of regular and rapid-acting insulin products have been limited by prolongation of PK/PD as the concentration of self-assembled insulin or insulin analogue is made higher than ca. 0.6 mM.
The present invention circumvents the necessity for insulin self-assembly as a mechanism to achieve a formulation of sufficient chemical stability and of sufficient physical stability to meet or exceed regulatory standards. Chemical degradation refers to changes in the arrangement of atoms in the insulin molecule, such as deamidation of Asn, formation of iso-Asp, and breakage of disulfide bridges. The susceptibility of insulin to chemical degradation is correlated with its thermodynamic stability (as probed by chemical denaturation experiments); because it is the monomer that is the species most susceptible to chemical degradation, its rate is reduced by sequestration of monomers within self-assemblies. Physical degradation refers to fibril formation (fibrillation), which is a non-native form of self-assembly that leads to linear structures containing thousands (or more) of insulin protomers in a beta-sheet rich conformation. Fibrillation is a serious concern in the manufacture, storage and use of insulin and insulin analogues above room temperature. Rates of fibrillation are enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, patients with DM optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for such patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus, and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable fluctuations in blood glucose levels or even dangerous hyperglycemia. At least one recent report has indicated that insulin Lispro (KP-insulin, an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; trade name Humalog®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters. Insulin exhibits an increase in degradation rate of 10-fold or more for each 10° C. increment in temperature above 25° C.; accordingly, guidelines call for storage at temperatures <30° C. and preferably with refrigeration. Such formulations typically include a predominance of native insulin self-assemblies.
The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable; in particular the lag time observed prior to onset of detectable fibrillation does not correlate with measurements of the thermodynamic stability of the native-state monomer (as probed by chemical denaturation experiments). Whereas a given substitution may stabilize both the overall native state and amyloidogenic partial fold—and so delay the onset of fibrillation—another substitution may stabilize the native state but not the amyloidogenic partial fold and so have little or no effect on the lag time. Still other substitutions may destabilize the native state but stabilize the amyloidogenic partial fold, and so lead to accelerated fibrillation despite its apparent stabilizing properties.
There is a need, therefore for an insulin analogue that displays rapid PK/PD for the treatment of DM under a broad range of insulin concentrations from 0.6 mM to 3.0 mM (typically corresponding to formulation strengths in a range from U-100 to U-500) while exhibiting at least a portion of the activity of the corresponding wild-type insulin, maintaining at least a portion of its chemical and/or physical stability.